Mol Biotechnol (2009) 42:128–133
DOI 10.1007/s12033-009-9145-0
RESEARCH
Novel Strategies to Construct Complex Synthetic Vectors
to Produce DNA Molecular Weight Standards
Zhe Chen Æ Jianbing Wu Æ Xiaojuan Li Æ
Chunjiang Ye Æ He Wenxing
Published online: 22 January 2009
Ó Humana Press 2009
Abstract DNA molecular weight standards (DNA markers, nucleic acid ladders) are commonly used in molecular
biology laboratories as references to estimate the size of
various DNA samples in electrophoresis process. One
method of DNA marker production is digestion of synthetic
vectors harboring multiple DNA fragments of known sizes
by restriction enzymes. In this article, we described three
novel strategies—sequential DNA fragment ligation,
screening of ligation products by polymerase chain reaction
(PCR) with end primers, and ‘‘small fragment accumulation’’—for constructing complex synthetic vectors and
minimizing the mass differences between DNA fragments
produced from restrictive digestion of synthetic vectors.
The strategy could be applied to construct various complex
synthetic vectors to produce any type of low-range DNA
Zhe Chen and Jianbing Wu contributed to this work equally.
Z. Chen X. Li
National Clinical Research Base of Traditional Chinese
Medicine, Zhejiang Hospital of Traditional Chinese Medicine,
Zhejiang Chinese Medical University, Hangzhou 310006,
People’s Republic of China
J. Wu
Institute of Biotechnology, Zhejiang University, Hangzhou
310029, People’s Republic of China
C. Ye (&) H. Wenxing
Department of Biotechnology, College of Medicine and Life
Sciences, University of Jinan, Jinan 250022, People’s Republic
of China
e-mail: [email protected]
C. Ye
State Key Laboratory of Microbial Technology, College of Life
Science, Shandong University, Jinan 250100, People’s Republic
of China
markers, usually available commercially. In addition, the
strategy is useful for single-step ligation of multiple DNA
fragments for construction of complex synthetic vectors and
other applications in molecular biology field.
Keywords Complex synthetic vectors Molecular weight
standards PCR screening of ligation products Sequential ligation End primer
Introduction
DNA molecular weight standards are a mixture of DNA
fragments (double strands or single strands) of known
sizes. They are usually used in electrophoresis with DNA
samples in the neighboring lanes to serve as references and
help researchers estimate the sizes of DNA samples [1–4].
DNA markers can be produced by several methods, which
can be PCR products composed of multiple DNA molecules of known sizes or produced by digestion of synthetic
vectors (plasmids) isolated from Escherichia coli or specific genomic DNAs such as those of the lambda phage and
the mealworm beetle Tenebrio molitor, a recently added
source for producing low-range DNA markers [5]. The
advantage of digestion of synthetic vectors is that DNA
markers can be generated on an industrial scale through
E. coli fermentation with simple and mature processing
techniques. Moreover, it is a more practical process than
PCR amplification which is much more labor intensive on a
small scale as discussed previously [1]. E. coli fermentation for plasmid production is, in fact, large-scale (up to
50 L or more) in situ amplification of the target sequences
(the amplicon or replicon is the whole synthetic vector or
plasmid), which is catalyzed by native un-isolated bacterial
DNA polymerase in oscillators or fermentors, instead of
Mol Biotechnol (2009) 42:128–133
PCR machines which are mainly used for research instead
of production.
The challenging step in DNA marker production from
artificial vectors is the design and construction of complex
synthetic vectors, which, once constructed, can be used for
fermentation production conveniently and kept forever.
The other steps, such as fermentation of E. coli with target
plasmids, extraction and purification of recombinant plasmid, and restrictive digestion (complete or partial) with
specific enzyme(s), are routine and mature operations
[6, 7]. In contrast, DNA marker production from PCR
reactions requires chemically synthesized deoxyribonucleotide triphosphates (dNTPs) and primers, purified DNA
polymerase (such as Taq polymerase), template DNA, and
reaction buffer preparations, all of which render the process
highly labor intensive and expensive, and the reproducibility of PCR production is poor [1, 8].
The ideal complex synthetic vectors for DNA marker
production by E. coli fermentation should contain multiple
DNA fragments of defined sizes, which could be released
by simple digestion with restriction enzymes, of which
EcoR I is one of the cheapest and usually the best choice
[1]. Efforts to improve the synthetic vectors used in DNA
marker production usually involves the insertion of multiple DNA fragments into the vector and elimination of the
difference in molecular mass between large and small
DNA bands by increasing the copy number of the small
DNA fragments [1]. In this study, we reported three
strategies for cloning of multiple DNA fragments into a
single plasmid and the elimination of the difference in
molecular mass between long and short DNA fragments.
Materials and Methods
129
TaKaRa. Kits for DNA gel recovery, PCR fragment cloning, and plasmid extraction and purification were
purchased from Omega, Promega, and Qiagen, respectively. Taq polymerase, T4 ligase, calf intestinal alkaline
phosphatase (CIAP), and restriction enzymes (SphI, EcoRI,
PstI, SalI, and ApaI) are all ordered from TaKaRa. The
PCR primers were synthesized by Invitrogen.
pYE was a synthetic complex plasmid for the production
of 200-bp DNA ladder[1], which was further modified in
this study. V4, a 2 kb plasmid with only SphI and SalI
restriction sites, is derived from the pGEM-T vector by
sequence deletion.
Methods
Design of Lambda DNA Restriction Fragments
and PCR Primer Pairs
In order to demonstrate the strategy, we will construct a
complex vector containing 500-bp, 750-bp, 1,000-bp, and
2,000-bp DNA segments, which are the main DNA bands
(generated by PCR amplifications) of DL2 k, a popular DNA
marker in the market. For the simplicity and efficiency of
PCR reactions in this study, the lambda phage genomic DNA
[9] is chosen as the target PCR template, which is analyzed
using the DNAMAN software (http://www.lynnon.com) for
restrictive sites and for optimal primer sites. The selected
segment to be amplified did not contain any restriction sites
for EcoRI, SalI, SphI, and PstI as a part of the vector design,
and the primers were re-evaluated using the Primerselect
software (DNAstar package: http://www.dnastar.com) for
parameters that may affect the PCR amplification efficiency.
The selected primer pairs are listed in Table 1, some of
which harbor EcoRI or PstI restrictive sites as indicated.
Materials
PCR Amplification of Lambda DNA Fragments
Escherichia coli DH5a was used as the recipient strain for
high-frequency plasmid transformation. The lambda phage
genomic DNA used as PCR template was purchased from
The PCR reactions were performed in a 100-ll volume
containing 4U Taq DNA polymerase; 50 ng lambda phage
Table 1 Primer pairs designed using the DNAMAN and Primerselect for the construction of pUJN-1 and pUJN-2 complex synthetic vectors
from lambda phage genomic DNA
750 forward
50 - ACT GCT GGC GGC AAA TGA GCA G-30
750 reverse
50 -AG GAA TTC (EcoRI) ACG TAC TGT CCG GAA TAC AC-30
1000 forward
1000 reverse
50 -AG GAA TTC (EcoRI) CCG TGA GAG CTA TCC CTT CAC C-30
50 -AG GAA TTC (EcoRI) GTT CAT CTT TCG TCA TGG AC-30
500 forward
50 -AG GAA TTC (EcoRI) CCG TCG CAT CAT CAT GCA GA -30
500 reverse
50 -CCA GCA CCA TCG TGT TGT CC-30
750 forward-2
50 -AG CTG CAG (PstI) ACT GCT GGC GGC AAA TGA GCA G-30
Underlined bases are restrictive sites added to the complementary part of primers; the italics are protective bases for effective digestion with
restrictive enzymes
130
Mol Biotechnol (2009) 42:128–133
DNA template; 60 pmol of forward and reverse primers,
2.5 mM each of dATP, dCTP, dGTP, and dTTP; and 10 ll
10 9 PCR reaction buffer (500 mM KCl, 100 mM Tris–
HCl, 1% Triton X, 15 mM MgCl2; pH 8.8). DNA amplifications were carried out in a PTC-200 thermal controller
(MJ Research) with the preliminary denaturation step of
2 min at 95°C, followed by 30 cycles of 50 s at 94°C, 30 s at
55°C, and 0.5–1 min (determined by the length of the target
DNA fragment) at 72°C, and a final step of 5 min at 72°C.
Gel Purification of PCR-Amplified Lambda DNA
Fragments
The four PCR-amplified lambda DNA fragments (500 bp,
1,000 bp, 750 bp-1 from the primer ‘‘750 forward,’’ and
750 bp-2 from the primer ‘‘750 forward-2,’’ as listed in
Table 1) were electrophoresed on a 1% agarose gel under
low voltage (75 V) for 30 min and each PCR band was
excised under UV light and recovered using DNA gel
recovery kit (Omega) to increase the specificity and
improve the purity of DNA molecules.
Restriction Digestion and Sequential Ligation
of PCR-Amplified Lambda DNA Fragments
Then, all the four types of DNA fragments of 1 lg were
digested with EcoRI (30U) at 37°C for 3 h in a 100-ll
volume respectively. At the end of the digestion, the volume of digestion reaction was adjusted to 500 ll by adding
400 ll of TE buffer (10 mM Tris-HCl, 1 mM EDTA;
pH 8.0); an equal volume of phenol–chloroform was added
and mixed thoroughly and the mixture was centrifuged at
12,000 rpm for 5 min. The supernatant was extracted with
an equal volume of chloroform and centrifuged at
12,000 rpm for another 5 min. The EcoRI-digested DNA
molecules were precipitated by adding 60 ll of 3 M NaAc
(pH 5.2) and 600 ll isopropanol to the extracted
supernatant and mixing well; the mixture was maintained
at -20°C for 3 h.
The precipitated DNA fragments were collected by
centrifugation at 13,000 rpm for 5 min and dissolved in
50 ll ddH2O after being washed with 70% ethanol for
several times and dried. Two parallel ligation systems were
established with a mixture of EcoRI-digested 500-(100 ng)
and 1,000-bp (200 ng) fragments (molar ratio is about 1:1)
and T4 ligase (6U) at 16°C in a 30 ll volume for 2 h. Then,
the purified PCR products 750 bp-1(150 ng) and 750 bp2(150 ng) were added into the above two parallel ligation
systems respectively (sequential ligation) with ligation
buffer strength adjustment to be 40-ll volume, the two
ligation systems were transferred to 4°C incubator, and
kept overnight to achieve the highest ligation efficiency.
PCR Amplification to Isolate Desired Ligation Products
The ligation systems were transferred to 75°C water bath
and kept for 20 min to inactivate the T4 ligase, and the
ligation products (3 ll) were used as templates for the
following two PCR screening reactions: In the first reaction, the two end primers ‘‘750 forward’’ and ‘‘500
reverse’’ (Table 1) were used to amplify and screen the
pre-designed C1 ligation unit (Table 2); Similarly, in the
second PCR reaction, the two end primers ‘‘750 forward2’’ and ‘‘500 reverse’’ were used to amplify and screen the
pre-designed C2 ligation unit. For the above PCR screening
amplifications, the extension time at 72°C was set to
2.5 min to adapt to the 2.25-kb PCR template size.
Ta Cloning of Recombinant Lambda DNA PCR
Products
The PCR-amplified recombinant lambda DNA PCR products (C1 and C2) were purified and recovered with DNA
gel recovery kit and cloned into the pGEM-T Easy vector
Table 2 The construct and vector symbols used in the text and their molecular structure and characteristics
Construct or vector
symbol
Molecular structure and characteristics
C1
The 750-1000-500-bp ligation unit
C2
(PstI)-750-1000-500-bp ligation unit: C1 construct with 50 PstI restriction site
C3
(PstI-750-1000-500-bp(Pst I) unit: derived from C2 and released from V2 digested with PstI
C4
The (SphI)-750-1000-500-750-1000-500-bp-(SalI) unit released from V3 with SalI and SphI double digestion
V1
pGEM-T Easy harboring the C1 construct incorporated by TA cloning
V2
pGEM-T Easy harboring the C2 construct incorporated by TA cloning
V3(pUJN-1)
pGEM-T Easy harboring the C1 and C2 constructs by subcloning C2 into the V1
V4
A 2 kb vector derived from the pGEM-T Easy due to deletion of 1-kb fragment remaining in the SphI and SalI
restriction sites
V5(pUJN-2)
V4 vector harboring the C1 and C2 constructs(C4) transferred from the V3
Mol Biotechnol (2009) 42:128–133
131
Fig. 1 Restrictive sites for foreign DNA fragment integration in the
multi-cloning site (MCS) of pGEM-T Easy vector, where * indicates
the TA cloning site (for more details, refer to the Promega technical
manual No.042). The double EcoRI restriction sites and PstI site are
utilized and created by replicating and integrating at PstI sites as
described in the text
Fig. 2 Maps of the pUJN-1 plasmid (a) and pUJN-2 plasmid (b).
Specific DNA fragments (1000-bp, 500-bp, and 750-bp or (PstI) 750bp,) were amplified using PCR, purified, and ligated in a sequential
manner. The specific C1 and C2 ligation products were picked out by
PCR amplification using two corresponding end primers with the
ligation products as PCR template, and the amplified ligation units
were cloned into the pGEM-T Easy vector by TA cloning. The two
three-DNA-fragment units (C1 and C2) were integrated into a single
T vector (pUJN-1) to increase the copy number of DNA fragments.
Another plasmid—pUJN-2 (b)—was constructed by transferring the
six-DNA-fragment unit (C4) from the pUJN-1 into a 2-kb plasmid
vector (V4) at the SphI and SalI restriction sites. For details, see the
text and Table 2
by the TA cloning method according to the manufacturer’s
instructions (Promega technical manual No.042) (Fig. 1).
The resultant recombinant pGEM-T Easy vectors were
assigned as V1 and V2, respectively (Table 2).
orientations, in order to select plasmids with the required
orientation; the right border of pGEM-T Easy vector was
sequenced with the M13 reverse sequencing primer to
confirm the identities (500 or 750 bp) of the integrated
sequence.
Construction of pUJN-1
Subsequently, the C2 unit was excised from the V2 vector
with the restrictive enzyme PstI [one PstI site was from the
multi-cloning site (MCS) of the pGEM-T Easy vector and
the other was introduced by the ‘‘750 foward-2’’ primer]
(Fig. 1; Table 1). Simultaneously, the V1 vector was linearized with PstI and dephosphorylated with calf intestinal
alkaline phosphatase, and the newly formed C3 unit
(released from the PstI digestion of V2) (Fig. 1; Table 2)
was introduced into the V1 vector at the PstI site to form
the V3 vector (pUJN-1) (Fig. 2; Table 2). Theoretically,
C3 will be incorporated into V1 vector in two possible
Construction of pUJN-2
The 6-DNA fragment unite-C4 (Table 2) was excised out
pUJN-1 with SalI and SphI and transferred into V4, a
pGEM-T easy-derived 2-kb vector (author’s unpublished
data) containing SalI and SphI restriction sites; the newly
formed vector with C1 ? C2(C4) construct was assigned
as V5 (pUJN-2). So the only difference between V5
(pUJN-2) and V3 (pUJN-1) was the vector size.
Plasmid maps (pUJN-1 and pUJN-2) were depicted
using the PlasmidDraw software.
132
pYE-Derived Complex Synthetic Vectors to Minimize
the Difference of Molecular Mass
In order to demonstrate the third strategy further, a complex synthetic vector for the production of an 200-bp even
ladder, constructed previously in our lab [1], was modified
to generate a series of pYE4, pYE5, and pY7 by introducing 1-kb, 2-kb, and 4-kb DNA segments (which did not
contain any restriction sites for EcoRI) into the pYE vector
at the ApaI restriction site (Promega technical manual
No.042) to change the vector size into 4, 5, and 7 kb,
respectively.
Results and Discussion
Results
In this study, we described three novel strategies for efficient cloning of different multiple DNA fragments as
demonstrated in the construction of the two complex synthetic vectors (pUJN-1 and pUJN-2). Four types of DNA
fragments (500-bp, 750-bp-1, 750-bp-2, and 1000-bp) were
amplified from the lambda genomic DNA with specific
restriction sites introduced by primer design (Table 1). The
above DNA segments were digested with EcoRI and
sequentially ligated after purification by agarose gel electrophoresis. Theoretically, there were several different
ligation units: 750-500-bp, 750-1,000-bp, 1,000-500-bp,
and C1 (the target) units, of which only the 750-500-bp and
C1 ligation units could be amplified with PCR screening
using specific end primers; at the same time, the two
Fig. 3 The electrophoresis maps of the DNA ladders from EcoRIdigested pUJN-2 (a); and the combination of EcoRI-digested pUJN-1
and pUJN-2 with different loading amounts of DNA samples (b, c) to
show the minimized differences in the EB-binding capacity of DNA
fragments with different sizes by accumulating the copy number of
relatively small ones in the vectors
Mol Biotechnol (2009) 42:128–133
possible ligation units (750-500-bp and C1) could be differentiated by electrophoresis. The sequential ligation of
the three DNA fragments ensured the generation of the C1
and C2 constructs (Table 2). The amplified C1 and C2
constructs were introduced into T vectors to form the V1
and V2 recombinant plasmids harboring the C1 and C2
constructs, respectively, from which V3 (pUJN-1) containing six DNA fragments, was constructed by subcloning C2 construct into the V1 vector. The orientation of
C2 construct in pUJN-1 was determined by sequencing
with the M13 reverse-sequencing primer, and a single
reaction will reveal the identity of the adjacent DNA
fragment (750 bp or 500 bp), so the orientation. Results
showed that the majority of the recombinant vectors were
with the desired orientation of C2 as depicted in the
Fig. 2a. The integrated six-DNA-fragment unit (C4) was
transferred with SalI and SphI digestion into a 2-kb vector
(V4) that had been constructed previously to form the V5
vector (pUJN-2) (Fig. 2b).
Three pYE-derived complex synthetic vectors (pYE4,
pYE5, pYE7,) were generated by modifying the vector size
Fig. 4 The electrophoresis map of pYEm (four pYE-related complex
synthetic vectors: pYE, pYE4, pYE5, and pYE7) digested with
EcoRI, which differ only in the vector size with the same inserted
DNA fragments (a), three of which are derived from the pYE as
detailed in the text. The 200-bp ladder (the pYE lane in B) generated
from the original pYE plasmid (b) was previously reported [2], from
which three derived vectors were constructed by incorporating 1-kb,
2-kb, and 4-kb fragments into the vector sequence at the ApaI site,
resulting in the accumulation of small DNA inserts (2 kb, 1.4 kb,
1 kb, 800 bp, 600 bp, 400 bp) released after restrictive digestion to
minimize the difference in molecular mass. Contrary to the DNA
marker generated from pYE-related vectors, the most bands of Dl2-k
and 100-bp ladder were PCR products
Mol Biotechnol (2009) 42:128–133
of pYE [1], which have the same insertion DNA profile.
When digested with EcoRI and mixed together with proper
ratios, accumulated were the inserted small DNA fragments and not the large vector bands (Fig. 4a).
Discussion
Usually, multiple DNA fragments of different sizes were
cloned into target vectors independently and repeatedly,
which involves repeated PCR amplification, gel purification and recovery of the DNA fragments, ligation and
transformation followed by tedious molecular biologybased screening steps [1]. Methods for cloning multiple
copies of a specific DNA fragment (such as 100 bp) have
been reported [10] and applied in the construction of DNA
ladder producing vectors [2–4] that require partial restriction digestion to generate a series of DNA bands; the DNA
markers produced by this method are not flexible, as discussed previously [1].
In this study, the designed DNA fragments were ligated
in a sequential manner, which defined the first formation of
1,000-500-bp construct and then the formation of C1 or C2
construct (still the minority of the ligated products) with
the addition of 750-bp. The PCR amplification of the
ligation products of different DNA fragments had two
functions. The first is the screening and accumulation of
the desired C1 and C2 constructs from the sequential
ligation, which are the minority in the ligation products and
difficult to be picked up by other methods, such as transformation and plasmid checking; the second function is to
add an A (adenosine) tail to the PCR-amplified C1 and C2
constructs and clone them into vectors with T (thymine)
tails (T vectors) conveniently and directly. This is the first
study to report such a technical strategy for multiple DNA
fragment ligation and ligation product screening.
After complete digestion with EcoRI, the V3 vector
could release several DNA molecules with a single copy of
3 kb and two copies of 750-bp, 1000-bp, and 500-bp. On
the aragose gel, the 3-kb fragment was much brighter than
the other bands due to its greater capacity to bind ethidium
bromide (EB) which resulted from its larger molecular
mass, which is also the case for the most commercial DNA
ladders from biotechnical companies generated with
digestion, such as the ‘‘E-Gel Low Range Quantitative
DNA Ladder’’ and ‘‘E-Gel High Range Quantitative DNA
Ladder’’ from Invitrogen. To minimize this difference,
pUJN-2 was constructed by transferring the C4 into V4.
Thus, after restriction digestion with EcoRI, V3(pUJN-1)
and V5 (pUJN-2) could release 1 9 3-kb, 2 9 1-kb,
2 9 750-bp, 2 9 500-bp, and 1 9 2-kb, 2 9 1-kb,
133
2 9 750-bp, 2 9 500-bp DNA molecules respectively for
each V3 and V5 molecule, a mixture of which will lead to
the accumulation of DNA molecules of the same size and
the minimization of the differences in EB-binding capacity
among the vector bands and the other bands from inserted
DNA sequences due to the accumulative effects of small
DNA molecules (Fig. 3b, c). This strategy can be extended
further; for example, the integrated six-DNA-fragment unit
(C4) (Table 2) could be transferred into 2.5-kb, 3.5-kb, and
4-kb vectors to reinforce this effect of accumulation of
small fragments, as demonstrated in the pYE-derived
200-bp ladder example (Fig. 4a).
The novel strategies described in this study are helpful
in constructing complex synthetic vectors, not only for
yielding better and inexpensive home–made DNA markers
in an easier and more practical manner, but also for flexible
ligation of multiple DNA segments in a more efficient way.
Acknowledgement This work is financially supported by the
‘‘Initiative Fund for Ph.D Fellows’’ from University of Jinan (Fund
No. B0524) and the National Natural Science Foundation of China
(No. 30600280 and No. 30700561).
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